Differential role of pannexin-1/ATP/P2X7 axis in IL-1ß release by human monocytes.

IL-1ß release is integral to the innate immune system. The release of mature IL-1ß depends on 2 regulated events: the de novo induction of pro-IL-1ß, generally via NF-κB-dependent transduction pathways; and the assembly and activation of the NLRP3 inflammasome. This latter step is reliant on active caspase-1, pannexin-1, and P2X7 receptor activation. Pathogen-associated molecular patterns in gram-positive and gram-negative bacteria activate IL-1ß release from immune cells via TLR2 and TLR4 receptors, respectively. We found that pro-IL-1ß and mature IL-1ß release from human monocytes is stimulated by the TLR2 agonists Pam3CSK4 or FSL-1, as well as the TLR4 agonist LPS in the absence of additional ATP. TLR2 agonists required pannexin-1 and P2X7 receptor activation to stimulate IL-1ß release. In contrast, IL-1ß release stimulated by the TLR4 agonist LPS is independent of both pannexin-1 and P2X7 activation. In the absence of exogenous ATP, P2X7 activation requires endogenous ATP release, which occurs in some cells via pannexin-1. In line with this, we found that LPS-stimulated human monocytes released relatively low levels of ATP, whereas cells stimulated with TLR2 agonists released high levels of ATP. These findings suggest that in human monocytes, both TLR2 and TLR4 signaling induce pro-IL-1ß expression, but the mechanism by which they activate caspase-1 diverges at the level of the pannexin-1/ATP/P2X7 axis.-Parzych, K., Zetterqvist, A. V., Wright, W. R., Kirkby, N. S., Mitchell, J. A., Paul-Clark, M. J. Differential role of pannexin-1/ATP/P2X7 axis in IL-1ß release by human monocytes.

Protocol for a human in vivo model of acute cigarette smoke inhalation challenge in smokers with COPD: monitoring the nasal and systemic immune response using a network biology approach.

INTRODUCTION: Cigarette smoke contributes to a diverse range of diseases including chronic obstructive pulmonary disease (COPD), cardiovascular disorders and many cancers. There currently is a need for human challenge models, to assess the acute effects of a controlled cigarette smoke stimulus, followed by serial sampling of blood and respiratory tissue for advanced molecular profiling. We employ precision sampling of nasal mucosal lining fluid by absorption to permit soluble mediators measurement in eluates. Serial nasal curettage was used for transcriptomic analysis of mucosal tissue. METHODS AND ANALYSIS: Three groups of strictly defined patients will be studied: 12 smokers with COPD (GOLD Stage 2) with emphysema, 12 matched smokers with normal lung function and no evidence of emphysema, and 12 matched never smokers with normal spirometry. Patients in the smoking groups are current smokers, and will be given full support to stop smoking immediately after this study. In giving a controlled cigarette smoke stimulus, all patients will have abstained from smoking for 12 h, and will smoke two cigarettes with expiration through the nose in a ventilated chamber. Before and after inhalation of cigarette smoke, a series of samples will be taken from the blood, nasal mucosal lining fluid and nasal tissue by curettage. Analysis of plasma nicotine and metabolites in relation to levels of soluble inflammatory mediators in nasal lining fluid and blood, as well as assessing nasal transcriptomics, ex vivo blood platelet aggregation and leucocyte responses to toll-like receptor agonists will be undertaken. IMPLICATIONS: Development of acute cigarette smoke challenge models has promise for the study of molecular effects of smoking in a range of pathological processes. ETHICS AND DISSEMINATION: This study was approved by the West London National Research Ethics Committee (12/LO/1101). The study findings will be presented at conferences and will be reported in peer-reviewed journals.

COX-2 protects against atherosclerosis independently of local vascular prostacyclin: identification of COX-2 associated pathways implicate Rgl1 and lymphocyte networks.

Cyxlo-oxygenase (COX)-2 inhibitors, including traditional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with increased cardiovascular side effects, including myocardial infarction. We and others have shown that COX-1 and not COX-2 drives vascular prostacyclin in the healthy cardiovascular system, re-opening the question of how COX-2 might regulate cardiovascular health. In diseased, atherosclerotic vessels, the relative contribution of COX-2 to prostacyclin formation is not clear. Here we have used apoE(-/-)/COX-2(-/-) mice to show that, whilst COX-2 profoundly limits atherosclerosis, this protection is independent of local prostacyclin release. These data further illustrate the need to look for new explanations, targets and pathways to define the COX/NSAID/cardiovascular risk axis. Gene expression profiles in tissues from apoE(-/-)/COX-2(-/-) mice showed increased lymphocyte pathways that were validated by showing increased T-lymphocytes in plaques and elevated plasma Th1-type cytokines. In addition, we identified a novel target gene, rgl1, whose expression was strongly reduced by COX-2 deletion across all examined tissues. This study is the first to demonstrate that COX-2 protects vessels against atherosclerotic lesions independently of local vascular prostacyclin and uses systems biology approaches to identify new mechanisms relevant to development of next generation NSAIDs.

Cyclooxygenase and cytokine regulation in lung fibroblasts activated with viral versus bacterial pathogen associated molecular patterns.

Cyclooxygenase (COX) is required for prostanoid (e.g. prostaglandin PGE2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1(-/-) and COX-2(-/-) mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1-8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24h) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.

The peroxisome proliferator-activated receptor ß/δ agonist GW0742 has direct protective effects on right heart hypertrophy.

Pulmonary hypertension is a debilitating disease with no cure. We have previously shown that peroxisome proliferator-activated receptor (PPAR) ß/δ agonists protect the right heart in hypoxia-driven pulmonary hypertension without affecting vascular remodeling. PPARß/δ is an important receptor in lipid metabolism, athletic performance, and the sensing of prostacyclin. Treatment of right heart hypertrophy and failure in pulmonary hypertension is an emerging target for future therapy. Here we have investigated the potential of GW0742, a PPARß agonist, to act directly on the right heart in vivo and what transcriptomic signatures are associated with its actions. Right heart hypertrophy and failure was induced in mice using a pulmonary artery banding (PAB) model. GW0742 was administered throughout the study. Cardiovascular parameters were measured using echocardiography and pressure monitoring. Fibrosis and cellular changes were measured using immunohistochemistry. Transcriptomics were measured using the Illumina MouseRef-8v3 BeadChip array and analyzed using GeneSpring GX (ver. 11.0). PAB resulted in right heart hypertrophy and failure and in increased fibrosis. GW0742 reduced or prevented the effects of PAB on all parameters measured. GW0742 altered a number of genes in the transcriptome, with Angptl4 emerging as the top gene altered (increased) in animals with PAB. In conclusion, the PPARß/δ agonist GW0742 has direct protective effects on the right heart in vivo. These observations identify PPARß/δ as a viable therapeutic target to treat pulmonary hypertension that may complement current and future vasodilator drugs.

Viral Toll Like Receptor activation of pulmonary vascular smooth muscle cells results in endothelin-1 generation; relevance to pathogenesis of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal condition in which raised pulmonary vascular resistance leads to right heart failure and death. Endothelin-1 is a potent endogenous vasoconstrictor, which is considered to be central to many of the events that lead to PAH, and is an important therapeutic target in the treatment of the condition. In many cases of PAH, the aetiology is unknown but inflammation is increasingly thought to play an important role and viruses have been implicated in the development of disease. The Toll Like Receptors (TLRs) play a key role in innate immune responses by initiating specific anti-bacterial and anti-viral defences in recognition of signature molecular motifs on the surface of invading pathogens. In this study, we set out to examine the expression of bacterial and viral TLRs in human pulmonary artery smooth muscle cells and to establish whether their activation could be relevant to PAH. We found that the viral TLR3 and bacterial TLRs 4 and 6 were most abundantly expressed in human pulmonary artery smooth muscle cells. Using specific TLR ligands, we found that activation of TLRs 3 and 4 resulted in IL-8 release by human pulmonary artery smooth muscle cells but that only TLR3 stimulation resulted in IP10 and endothelin-1 release. These data suggest that human pulmonary artery smooth muscle cells express significant levels of viral TLR3 and respond to its activation by releasing endothelin-1. This may have importance in understanding the association between viruses and the development of PAH.

Inflammatory transcriptome profiling of human monocytes exposed acutely to cigarette smoke.

BACKGROUND: Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 10(15) free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases. METHODOLOGY/PRINCIPLE FINDINGS: To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway. CONCLUSIONS: Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes.

Antiplatelet actions of statins and fibrates are mediated by PPARs.

OBJECTIVES: Statins and fibrates are hypolipidemic drugs which decrease cardiac events in individuals without raised levels of cholesterol. These drugs inhibit platelet function, but the mechanisms by which this pleiotropic effect is exerted are not known. METHODS AND RESULTS: We used a range of approaches to show statins inhibit human platelet activation in vitro while engaging PPARalpha and PPARgamma. The effects of simvastatin were prevented by the PPARgamma antagonist GW9662 or the PPARalpha antagonist GW6471. In a small-scale human study fluvastatin activated PPARalpha and PPARgamma in platelets and reduced aggregation in response to arachidonic acid ex vivo. The effects of fenofibrate were prevented by PPARalpha antagonism with GW6471. Fenofibrate increased bleeding time in wild-type, but not in PPARalpha-/- mice. The inhibitory effect of fenofibrate, but not simvastatin, on aggregation was prevented by deletion of PPARalpha in murine platelets. PKCalpha, which influences platelet activation, associated and immune-precipitated with PPARgamma in platelets stimulated with statins and with PPARalpha in platelets stimulated with fenofibrate. CONCLUSIONS: This study is the first to provide a unifying explanation of how fibrates and statins reduce thrombotic and cardiovascular risk. Our findings that PPARs associate with PKCalpha in platelets also provide a mechanism by which these effects are mediated.

Toll-like receptor 2 is essential for the sensing of oxidants during inflammation.

RATIONALE: The mechanisms by which oxidants are sensed by cells and cause inflammation are not well understood. OBJECTIVES: This study aimed to determine how cells "sense" soluble oxidants and how this is translated into an inflammatory reaction. METHODS: Monocytes, macrophages, or HEK293 cells (stably transfected with human Toll-like receptor [TLR]2, TLR2/1, TLR2/6, or TLR4/MD2-CD14) were used. CXC ligand-8 (CXCL8) levels were measured using ELISA. Phosphorylated IL-1 receptor-associated kinase 1 levels were measured using Western blot. TLR2(-/-) and TLR4(-/-) mice were challenged with oxidants, and inflammation was measured by monitoring cell infiltration and KC levels. MEASUREMENTS AND MAIN RESULTS: Oxidants evoked the release of CXCL8 from monocytes/macrophages; this was abrogated by pretreatment with N-acetylcysteine or binding antibodies to TLR2 and was associated with the rapid phosphorylation of IL-1 receptor-associated kinase 1. Oxidants added to HEK293 cells transfected with TLR2, TLR1/2, or TLR2/6 but not TLR4/MD2-CD14 or control HEK nulls resulted in the release of CXCL8. Oxidant challenge delivered intraperitoneally (2-24 hours) or by inhalation to the lungs (3 days) resulted in a robust inflammation in wild-type mice. TLR2(-/-) mice did not respond to oxidant challenge in either model. TLR4(-/-) mice responded as wild-type mice to oxidants at 2 hours but as TLR2(-/-) mice at later time points. CONCLUSIONS: Oxidant-TLR2 interactions provide a signal that initiates the inflammatory response.

Gram-positive and Gram-negative bacteria synergize with oxidants to release CXCL8 from innate immune cells.

We have recently demonstrated that oxidants can activate monocytes via an action on Toll-like receptor (TLR) 2; however, it is unclear what functional consequence this has on immune surveillance for Gram-negative and -positive bacteria. Gram-negative and -positive bacteria and their related pathogen-associated molecular patterns (PAMPs) are sensed by TLR4 and TLR2, respectively. In the current study, we used a human monocyte cell line to show that oxidants prime cells to subsequent challenge with Gram-negative or -positive bacteria as well as PAMPs specific for TLR4 (LPS), TLR2/1 (Pam(3)CSK4), TLR2/6 (FSL-1), Nod1 (FK565), and Nod2 (MDP Lys 18). Similarly, activation of TLR4 with LPS primed for subsequent activation of cells by agonists of the TLR2/6 or TLR2/1 complex. However, no synergy was noted when cells were costimulated with Pam(3)CSK4 and FSL-1. We then tested blood (and isolated monocytes) derived from healthy smokers, which is oxidant primed, making it more sensitive to bacterial or PAMP stimulation when compared with blood of nonsmokers. Thus an oxidant stimulation, possibly via an action on TLR2 or associated transduction pathways, provides a signal that initiates inflammatory responses and sensitizes cells to pathogenic insults.

Critical role of toll-like receptors and nucleotide oligomerisation domain in the regulation of health and disease.

Pathogens are sensed by pattern recognition receptors (PRRs), which are germ line-encoded receptors, including transmembrane Toll-like receptors (TLRs) and cytosolic nucleotide oligomerisation domain (NOD) proteins, containing leucine-rich repeats (NLRs). Activation of PRRs by specific pathogen-associated molecular patterns (PAMPs) results in genomic responses in host cells involving activation transcription factors and the induction of genes. There are now at least 10 TLRs in humans and 13 in mice, and 2 NLRs (NOD1 and NOD2). TLR signalling is via interactions with adaptor proteins including MyD88 and toll-receptor associated activator of interferon (TRIF). NOD signalling is via the inflammasome and involves activation of Rip-like interactive clarp kinase (RICK). Bacterial lipopolysaccharide (LPS) from Gram-negative bacteria is the best-studied PAMP and is activated by or 'sensed' by TLR4. Lipoteichoic acid (LTA) from Gram-positive bacteria is sensed by TLR2. TLR4 and TLR2 have different signalling cascades, although activation of either results in symptoms of sepsis and shock. This review describes the rapidly expanding field of pathogen-sensing receptors and uses LPS and LTA as examples of how these pathways parallel and diverge from each other. The role of pathogen-sensing pathways in disease is also discussed.

Calcitonin and prednisolone display antagonistic actions on bone and have synergistic effects in experimental arthritis.

We tested here the hypothesis that calcitonin and glucocorticoids, known to modulate bone metabolism, could have opposite actions on bone cells regulating expression of cytokine receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG). In the U2OS osteosarcoma cell line, calcitonin (10(-11) to 10(-9) mol/L) reduced RANKL and augmented OPG both at the mRNA and protein levels. Cell incubation with prednisolone (10(-8) to 10(-6) mol/L), the glucocorticoid chosen for this study, produced opposite results. These molecular studies prompted more functional analyses whereby osteoclast bone resorptive activity was determined. Calcitonin (10(-10) mol/L) abrogated the stimulating effect of 10 ng/ml RANKL or 10(-9) mol/L prednisolone; similar results were obtained with OPG. Assessment of calcitonin and prednisolone effects in an in vivo model of rheumatoid arthritis revealed partially surprising results. In fact, calcitonin not only preserved bone morphology (as assessed on day 18) in rats subjected to arthritis and treated with prednisolone (0.8 to 4 mg/kg daily from day 13) but also synergized with the steroid to elicit its antiarthritic effects. These results suggest that calcitonin could be used as a novel cotreatment to augment efficacy and reduce side effects associated with the prolonged use of steroids.

Selective NOD1 agonists cause shock and organ injury/dysfunction in vivo.

RATIONALE: NLRs (nucleotide oligomerisation domain [NOD] proteins containing a leucine-rich repeat) are cytosolic pattern recognition receptors. NOD1 senses diaminopimelic acid-containing peptidoglycan present in gram-negative bacteria, whereas NOD2 senses the muramyl dipeptide (MDP) present in most organisms. Bacteria are the most common cause of septic shock, which is characterized clinically by hypotension resistant to vasopressor agents. In animal models, gram-negative septic shock is mimicked by lipopolysaccharide (LPS), which signals through Toll-like receptor 4 (TLR4) and its adaptor MyD88. The role of NLRs in the pathophysiology of septic shock is not known. OBJECTIVES: To compare the effects of selective NOD1 agonists with LPS in vivo. METHODS: Vascular smooth muscle cells or whole aortas from wild-type or genetically modified mice were stimulated in vitro with agonists of NOD1 (FK565) or NOD2 (MDP). Vasoconstriction was measured using wire myography. Nitric oxide (NO) formation was measured using Griess reaction and NO synthase-II protein by Western blotting. In vivo, blood pressure, heart rate, and urine output were measured in sham-, LPS-, or FK565-treated animals. Biomarkers of end-organ injury, coagulation activation, NO, and cytokines were measured in plasma. MAIN RESULTS: FK565, but not MDP, induced NO synthase-II protein/activity in vascular smooth muscle and vascular hyporeactivity to pressor agents. FK565 had no effect on vessels from NOD1(-/-) mice, but was active in vessels from TLR4(-/-), TLR2(-/-), or MyD88(-/-) mice. FK565 induced hypotension, increased heart rate, and caused multiple (renal, liver) injury and dysfunction in vivo. CONCLUSIONS: Activation of NOD1 induces shock and multiple organ injury/dysfunction.

Differential effects of Gram-positive versus Gram-negative bacteria on NOSII and TNFalpha in macrophages: role of TLRs in synergy between the two.

1. Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFalpha are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFalpha induction. 2. In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFalpha release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam(3)CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFalpha without an effect on NO. 3. At later time points (48-72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFalpha-binding antibodies. 4. At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. 5. Using cells from TLR2(-/-) or TLR4(-/-) mice, the ability of LPS to synergise with S. aureus or Pam(3)CSK4 was found to be dependent on both TLR2 and TLR4. 6. These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFalpha genes compared with their coinduction when both receptor pathways are activated.

Prostaglandin F2alpha produced by inducible cyclooxygenase may contribute to the resolution of inflammation.

Cyclooxygenase-2 may play a role in resolution of carrageenan-induced pleurisy in rats by generating anti-inflammatory prostanoids. Here, we show exudate prostaglandin F2alpha concentrations rise during resolution of this model. These were reduced by the selective cyclooxygenase-2 inhibitor NS-398, which exacerbated inflammation. Concomitant treatment with NS-398 and the synthetic FP receptor agonist fluprostenol reversed this exacerbation. This suggests prostaglandin F2alpha produced by cyclooxygenase-2 contributes to resolution of this inflammatory reaction.

Ligand-specific glucocorticoid receptor activation in human platelets.

Few studies have addressed the effects of classical anti-inflammatory glucocorticoids on platelet function. Here, we report for the first time that human platelets contain the glucocorticoid receptor (GR) as identified by a combination of biochemical and functional techniques. Ligand-binding studies revealed the presence of a high- and low-affinity binding site for [3H]-dexamethasone in platelets. The 2 GR ligands prednisolone and dexamethasone competed for [3H]-dexamethasone binding, as did the mineralocorticoid aldosterone. However, while prednisolone (1-10 microM) reduced adenosine diphosphate (ADP, 4 microM) and thromboxane A2 receptor agonist U46619 induced platelet aggregation (up to 75%), dexamethasone had no effect. The inhibition produced by prednisolone was reversed by preincubation with the GR antagonist mifepristone (10 microM; RU486), suggesting the functional importance of the ligand-receptor complex. In addition, prednisolone caused a marked (approximately 50%) reduction in thromboxane B2 levels, whereas dexamethasone was without effect. The apparently anomalous binding data were clarified by the fact that washed platelets (1) contained mineralocorticoid receptor and that (2) it was associated with GR. Taken together, our data suggest that platelet GR forms a heterodimeric complex with the mineralocorticoid receptor that is susceptible to differential activation by specific receptor ligands.

Cigarette smoke activates human monocytes by an oxidant-AP-1 signaling pathway: implications for steroid resistance.

Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smoking-induced pathophysiology is often resistant to the anti-inflammatory effects of glucocorticoids. The nature of cigarette smoke-induced inflammation is still not defined, although neutrophil recruitment and activation seem to be consistent features. In the current study, we have used a range of approaches to demonstrate that cigarette smoke activates human monocytes and macrophages to release the CXC chemokine CXCL8 [(interleukin-8 (IL-8)]. Furthermore, we show for the first time that cigarette smoke synergizes with proinflammatory cytokines IL-1beta and tumor necrosis factor-alpha, and it is this interaction that confers steroid resistance to smoke-induced CXCL8 release. We go on to show that smoke-induced activation of human cells is an oxidant-mediated phenomenon acting through activator protein-1, but not nuclear factor kappaB, pathway. These observations add significantly to our understanding of smoke as an inflammatory stimulus that has implications for potential the development of treatments of smoking or related disease.

15-epi-lipoxin A4-mediated induction of nitric oxide explains how aspirin inhibits acute inflammation.

The established model for the mechanism of action of aspirin is the inhibition of prostaglandin synthesis. However, this has never fully explained aspirin's repertoire of antiinflammatory properties. We found in acute pleuritis that aspirin, but not salicylate, indomethacin, or piroxicam, increased plasma nitric oxide (NO), which correlated with a reduction in inflammation. Inhibiting aspirin-elicited NO pharmacologically in this model nullified the antiinflammatory effects of aspirin. Moreover, aspirin was not antiinflammatory in either constitutive (eNOS) or inducible NO synthase (iNOS) knockout mice with IL-1beta-induced peritonitis. It transpires that aspirin generates NO through its unique ability to trigger the synthesis of 15-epi-lipoxin A(4). Aspirin and 15-epi-lipoxin A(4) were shown to inhibit leukocyte trafficking in an NO-dependent manner using intravital microscopy on IL-1beta-stimulated mouse mesentery. Not only did aspirin inhibit leukocyte-endothelial interaction in a manner similar to NO in wild-type mice but both aspirin and 15-epi-lipoxin A(4) had markedly reduced effects on leukocyte-endothelial cell adherence in eNOS- and iNOS-deficient mice compared with wild type. Collectively, these data suggest that aspirin triggers the synthesis of 15-epi-lipoxin A(4), which increases NO synthesis through eNOS and iNOS. This aspirin-elicited NO exerts antiinflammatory effects in the microcirculation by inhibiting leukocyte-endothelium interactions.

An isocratic HPLC method for the quantitation of eicosanoids in human platelets.

We describe here a modified protocol for the simultaneous quantification of specific eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B(2) (TXB(2)), arachidonic acid (AA), 12-R-hydroxyeicosatetraenoic acid (12-R-HETE), 12-S-hydroxyheptadecatrienoic acid (12-S-HHTrE) and the internal standard prostaglandin B(1) (PGB(1)) were extracted from human platelets by liquid-liquid extraction using ethyl acetate. This was followed by derivatization and fluorescent detection prior to analysis by reversed phase liquid chromatography. The high-performance liquid chromatographic method consisted of ODS reversed-phase column (3 microm) and a mobile phase of acetonitrile-water (85:15). TXB(2) and AA plasma calibration curves were linear between 6.25 and 125 ng mL(-1) (r(2) > 0.997), whereas for 12-R-HETE and 12-S-HHTrE the curves were linear between 5.0 and 40 ng mL(-1) (r(2) > 0.998). All calibration curve standards had <15% CV (coefficient of variation) and between-run precision, and the percentage relative deviation for replicate (n = 6) quality controls was less than 5.5%. The method was adapted to allow the screening of drugs that may affect either one or both of the lipoxygenase and cyclo-oxygenase pathways.

Synthesis of nitro esters of prednisolone, new compounds combining pharmacological properties of both glucocorticoids and nitric oxide.

Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development.